Mechanism and Regulation of Phage Mu Transposition The ubiquitous presence of transposable elements has contributed to genome evolution on the one hand, and genome instability on the other, the latter contributing to human disease, especially by oncogenic retroviruses. The integration mechanism of our model virus Mu is remarkably similar to that of a retrovirus. Retroviral integration is followed by DNA repair, while Mu integration has a choice between repair and replication, depending on the phase of its life cycle. Despite extensive research, answers to three important questions in Mu biology, which are intimately related to retroviral integration, have remained elusive. These are: (1) the regulatory decision between repair and replication of a common transposition intermediate, (2) the mechanism by which Mu avoids integrating into itself, and (3) how Mu chooses target sites in vivo. In the present grant cycle, a new window was opened into all three questions by several discoveries - a DNA repair function in the transposase itself, a retroviral BAF-like mechanism we call 'Mu genome immunity' which employs MuB, and a target selection process that senses chromosome domain structure. The specific aims of this proposal will address the following questions: 1. Why does a common 8 strand transfer intermediate proceed down the repair pathway upon Mu infection, but replicative pathway during lytic growth? The repair pathway is hitherto unexplored. We have discovered that the cryptic endonuclease activity within the C-terminal domain of MuA is required for repair in vivo. Also required in vivo is the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. An additional requirement is transcription from a promoter that lies within the enhancer, which is an integral part of the transpososome. We hypothesize that ClpX constitutes part of highly regulated mechanism that unmasks the nuclease activity of MuA under early transcription conditions during infection, and propose tests for this hypothesis. 2. How does MuB participate in 'Mu genome-immunity'? Two rival immunity mechanisms - genome immunity and cis-immunity - operate inside and outside Mu ends, respectively, suggesting that Mu is segregated into an independent chromosomal domain. We propose experiments to understand Mu domain structure and Mu immunity. 3. How does MuB influence target site selection in vivo? Our results suggest that Mu integration is responsive to some aspect of chromosome structure that controls MuB binding. We propose to test the influence of several cellular nucleoid-organizing proteins on target selection, and to extend these E. coli results to mammalian cells to determine how a eukaryotic chromosomal domain responds to MuB binding and Mu integration. These studies may eventually lead to the design of site-specific gene delivery vectors. PUBLIC HEALTH RELEVANCE: The long term goal of this research is to understand the mechanisms underlying the genetic success of transposable elements and to harness this knowledge to improve human health. The insights gained from the proposed studies will particularly impact our understanding of retroviral integration biology. They are also expected to lead to the design of site-specific gene delivery vectors.